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Franc Laboratory Research

Statement of Research Interest

Our laboratory is interested in genetically dissecting the molecular mechanisms of phagocytosis by macrophages, using Drosophila melanogaster as a model system.  Although we are mainly focussing on studying apoptotic cell clearance during embryogenesis, genes involved in this process may also be involved in bacterial phagocytosis during innate immune responses.  Our laboratory uses Drosophila genetics, classical molecular and cell biology tools, as well as classical, confocal, time-lapse and electron microscopy techniques.  Our long-term goal is to compare and contrast the molecular mechanisms associated with phagocytosis of apoptotic cells with that of pathogens, such as bacteria.  These studies are likely to shed light on the molecular mechanisms of phagocytosis in general, as well as how the specificity of recognition and engulfment of various particles might be achieved.  Failure to clear apoptotic cells has been associated with autoimmune diseases, such as Lupus Erythematosus, and may contribute to neurogenerative disorders.  Our hope is that our studies will increase our basic understanding of phagocytosis, an important aspect of the innate cellular immune response; and ultimately contribute to the design of new drugs to treat diseases associated with failed apoptotic cell clearance, as well as bacterial infections.  

 

Innate Immunity

Innate immunity provides the first line of host defence in all multi-cellular organisms. It relies on both humoral and cellular responses controlled by specialized immune cells and designed to limit the host infection by other organisms. Drosophila melanogaster has served as a particularly useful model organism to genetically dissect the humoral aspect of innate immunity. The immense knowledge derived from such studies led to the important discovery of the Toll Receptors and their signalling pathways in mammalian systems. To date, however, little is known still about the molecular mechanisms underlying the cellular aspect of innate immunity, such as phagocytosis.

Phagocytosis is the process by which specialized cells, namely phagocytes, recognize and engulf particles such as pathogens or tissue debris that accumulate during infection and wound repair. This process is critical for the survival of multicellular organisms, as it limits infection. Phagocytosis is also important during development, as it participates in the clearance of cells that undergo programmed cell death, such as apoptosis. Recent data suggest that failure to dispose of apoptotic cells contributes to autoimmune diseases such as Lupus erythematosus, and may contribute to neurodegenerative diseases. Moreover, phagocytosis of apoptotic cells triggers anti-inflammatory signals, which are critical in the resolution of inflammation.

Figure 1: A confocal image of a wild-type Drosophila embryo where macrophages express a UAS-eGFP transgene under the control of a crq-Gal4 driver. This embryo was fixed and stained with GFP antibody (green) Croquemort antibody (blue) and 7-Amino Actinomycin-D, a nuclear DNA dye that labels all nuclei and the condensed DNA of apoptotic corpses). Croquemort is a CD36-related Drosophila macrophage receptor that is required for efficient phagocytosis of apoptotic cells in vivo (Franc et al. 1996, Immunity 4:431-443). A close-up view at a single macrophage is shown in figure 2.

Figure 2: A confocal image of a wild-type Drosophila embryonic macrophage stained with Peroxidasin antibody (green) and Croquemort antibody (blue) that has engulfed 5 apoptotic corpses (bright red round particles stained with 7-Amino Actinomycin-D, a DNA dye). Peroxidasin is an extracellular matrix protein, which serves as a macrophage marker (Nelson et al. 1994. EMBO J.; Aug 1;13(15):3438-47). Croquemort is a CD36-related Drosophila macrophage receptor that is required for efficient phagocytosis of apoptotic cells in vivo (Franc et al. 1996, Immunity 4:431-443).

 

Our Research

To identify new components of the Drosophila macrophages phagocytic machinery, we carried out an in vivo genetic screen for deletion mutants with defect in apoptotic cell clearance by embryonic macrophages. Acridine Orange (AO), a nucleic acid selective fluorescent cationic dye was previously shown to stain all apoptotic cells in the fly embryo (Abrams et al., 1993). We observed that AO-stained apoptotic corpses were clustered in wild-type embryos, each cluster corresponding to individual macrophages having engulfed multiple apoptotic cells, while these clusters were lacking in crq-deletion mutant embryos that are deficient in apoptotic cell clearance (Silva et al. 2007, Immunity 4:541-4). Thus, we used AO staining to detect apoptotic cell clearance defects in Drosophila mutant embryos and identified several deletion mutants with defects in macrophage formation, proliferation, differentiation and/or maturation, migration, and phagocytic function (Franc NC and White K. unpublished data).

In our laboratory, we have been pursuing the characterization of the deletion mutants with defective apoptotic cell clearance, and used three strategies to identify the genes responsible for their phenotype:

1) Screen available P-element insertions or chemical mutants in the region of a deficiency of interest to identify potential single gene mutants with apoptotic cell clearance phenotype that may recapitulate that of the deletion mutant.

Using this approach, we recently identified pallbearer, a gene encoding a novel Drosophila F-Box protein as essential for efficient phagocytosis of apoptotic cells by embryonic macrophages (Silva et al. 2007, Immunity 4:541-4). (Click here to learn more about the Pallbearer Project)

2) Screen genes in the region of a deficiency of interest for their potential role in phagocytosis by knocking them down using RNA interference (RNAi) in S2 cells and testing in an in vitro phagocytosis assay.

Using this approach, we recently identified undertaker, a gene encoding a Junctophilin-related protein, as essential for efficient phagocytosis and calcium homeostasis (Cuttell et al. 2008, Cell; 135 (3):524-34). Click to learn more about the Undertaker Project

3) Study genes in the region of a deficiency of interest that have homologies with genes known to be involved in phagocytosis in other model systems (candidate genes approach).

Using this approach, we have been studying the role of the Drosophila homologue of Ced-12 (known as Elmo in mammals) in phagocytosis, and recently found a nearby gene, as being also involved in calcium homeostasis during phagocytosis (Van Goethem E., Bakatselou C., Silva E & Franc NC, manuscript in preparation).

Our research will continue along four lines:

1) Study the role of Pallbearer, ubiquitylation and proteasomal degradation in phagocytosis
2) Study the role of Undertaker and calcium homeostasis in phagocytosis
3) Study the in vivo dynamics of, and relationship between, all the molecules known to be involved in phagocytosis in the fly embryo
4) Pursue the genetic dissection of the molecular mechanisms of phagocytosis, and continue to highlight the similarities and differences in the molecular mechanisms of phagocytosis of apoptotic cells versus bacteria.

 

Selected Publications

Cuttell L*, Vaughan A*, Silva E, Escaron CE, Lavine M, van Goethem E, Eid J-P, Quirin M, Franc NC. Undertaker, a Drosophila Junctophilin links Draper-mediated Phagocytosis and Calcium Homeostasis. Cell. 2008; 135(3):524-34.  *These authors contributed equally to this work.

Silva EA, Burden J, Franc NC. In vivo and in vitro methods for studying apoptotic cell engulfment in Drosophila. Methods Enzymol. 2008;446:39-59. PMID: 18603115 [Medline Abstract]

Silva E*, Au-Yeung HW*, Van Goethem E, Burden J, Franc NC. Requirement for a Drosophila E3-ubiquitin ligase in phagocytosis of apoptotic cells.  Immunity. 2007 Oct;27(4):585-96. Epub 2007 Oct 11. PMID: 17936033 [MedlineAbstract]  *These authors contributed equally to this work.
Comments in: Immunity. 2007 Oct;27(4):541-4. [Medline Abstract]
Reviewed in: Trends Cell Biol. 2008 Mar;18(3):95-7. Epub 2008 Feb 15. [Medline Abstract]

Franc NC.  Phagocytosis of apoptotic cells in mammals, Caenorhabditis elegans and Drosophila melanogaster: molecular mechanisms and physiological consequences. Front Biosci. 2002 May 1;7:d1298-313. Review. PMID: 11991836 [Medline Abstract]

Franc NC, Heitzler P, Ezekowitz RA, White K. Requirement for croquemort in phagocytosis of apoptotic cells in Drosophila. Science. 1999 Jun 18;284(5422):1991-4. PMID: 10373118 [Medline Abstract]

Franc NC, Dimarcq JL, Lagueux M, Hoffmann J, Ezekowitz RA. Croquemort, a novel Drosophila hemocyte/macrophage receptor that recognizes apoptotic cells. Immunity. 1996 May;4(5):431-43. PMID: 8630729 [Medline Abstract]